Review γδ T Cells and Their Potential for · PDF fileγδ T Cells and...

Int. J. Biol. Sci. 2014, Vol. 10

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IInntteerrnnaattiioonnaall JJoouurrnnaall ooff BBiioollooggiiccaall SScciieenncceess 2014; 10(2):119-135. doi: 10.7150/ijbs.7823

Review

γδ T Cells and Their Potential for Immunotherapy Yan-Ling Wu1, Yan-Ping Ding1,2, Yoshimasa Tanaka3, Li-Wen Shen2, Chuan-He Wei2, Nagahiro Minato4, and Wen Zhang2

1. Lab of Molecular Immunology, Zhejiang Provincial Center for Disease Control and Prevention, 630 Xincheng Road, Hangzhou, 310051, China 2. Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road,

Hangzhou, 310014, China 3. Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501,

Japan 4. Department of Immunology and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan

Corresponding authors: Yanling Wu, Lab of Molecular Immunology, Zhejiang Provincial Center for Disease Control and Prevention, 630 Xincheng Road, Hangzhou, 310051, China; Tel: +86-571-87115282; Fax: +86-571-87115282; e-mail: [email protected]; Wen Zhang, Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, China; Tel: +86-571-88871507; Fax: +86-571-88871507; e-mail: [email protected].

© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.

Received: 2013.10.04; Accepted: 2013.12.17; Published: 2014.01.10

Abstract

Vγ9Vδ2 (also termed Vγ2Vδ2) T cells, a major human peripheral blood γδ T cell subset, recognize microbial (E)-4-hydroxy-3-methylbut-2-enyl diphosphate and endogenous isopentenyl diphos-phate in a TCR-dependent manner. The recognition does not require specific accessory cells, antigen uptake, antigen processing, or MHC class I, class II, or class Ib expression. This subset of T cells plays important roles in mediating innate immunity against a wide variety of infections and displays potent and broad cytotoxic activity against human tumor cells. Because γδT cells express both natural killer receptors such as NKG2D and γδ T cell receptors, they are considered to represent a link between innate and adaptive immunity. In addition, activated γδ T cells express a high level of antigen-presenting cell-related molecules and can present peptide antigens derived from destructed cells to αβ T cells. Utilizing these antimicrobial and anti-tumor properties of γδ T cells, preclinical and clinical trials have been conducted to develop novel immunotherapies for infections and malignancies. Here, we review the immunological properties of γδ T cells including the underlying recognition mechanism of nonpeptitde antigens and summarize the results of γδ T cell–based therapies so far performed. Based on the results of the reported trials, γδ T cells appear to be a promising tool for novel immunotherapies against certain types of diseases.

Key words: γδ T cells, nonpeptide antigen, tumor, infection, autoimmune and allergic diseases, immunotherapy

Introduction T cells are subdivided into two major popula-

tions distinguished by their surface expression of αβ and γδ T cell receptors (TCR). Both αβ and γδ T cells arise from common multipotent double negative (DN) precursors in the thymus, which can be further sepa-rated into four DN subsets based on CD44 and CD25 expression [1, 2]. The T cells undergo extensive DNA rearrangements at the β, γ and δ TCR loci aiming to express functional TCR chains and make a selection between two developmental programs during the

DN3 stage, thus generating two distinct characteris-tics and functions of T cell subsets [3, 4]. Cells with the αβ TCR generally express CD4 or CD8 lineage mark-ers and mostly fall into helper or cytotoxic/effector subsets, whereas, cells with the γδ TCR in humans usually do not express the lineage markers. They do not require conventional antigen presentation in the context of MHC [5]. In fact, the αβ and γδ T cell pop-ulations recognize different types of antigens. αβ T cells recognize non-self peptide fragments restricted

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by MHC molecules. γδ T cells, on the other hand, recognize unconventional antigens including stress molecules like MICA and MICB and non-peptidic metabolites of isoprenoid biosynthesis [6-9] and so on. Ligand recognition by γδ T cells appears to be dis-pensable for the selection of γδ T cells in the adult thymus [10].

Human γδ T cells can be divided into three main populations based on δ chain expression. γδ T cells expressing Vδ1 chains are prominent in the intraepi-thelial layer of mucosal surfaces, where they are in-volved in maintaining epithelial tissue integrity when facing damage, infection, or transformation [11], re-sponding to stress antigens on epithelial cells [12] and producing IL-10 but little or no IL-2, IL-4 or IFN-γ [13]. They also appear in the peripheral blood, but the intraepithelial and blood populations seem to mix little or not at all [14, 15]. The second population of γδ T cells expresses Vδ2 chain products and represents the majority of circulating γδ T lymphocytes in healthy human adults, comprising up to 50%–90% of the peripheral γδ T-cell population. The Vδ2 chain pairs almost exclusively with Vγ9 (also termed Vγ2). The Vγ9Vδ2 pairing is present only in humans and nonhuman primates [16]. One attractive feature of Vδ2 T cells is that they can serve as professional anti-gen-presenting cells (APCs) [17]. Activated Vδ2 T cells acquire characteristics of professional APC, such as the expression of antigen presenting, costimulatory, and adhesion molecules, including MHC-II, CD80, and CD86 [18]. It is worthy of note that rapamycin or IL-18 treatment is reported to increase the expression of MHC-II, CD80, and CD86 on Vδ2 T cell lines [18]. The third population is Vδ3 T cells, which take up about 0.2% of circulating T cells including CD4+, CD8+, and CD4-CD8- subsets. They are expressed CD56, CD161, HLA-DR, and NKG2D but without NKG2A and NKG2C [19]. The Vδ3 T cells are found only a little in blood but are rich in liver and in pa-tients with leukemias and some chronic viral infec-tions. Through mitogen stimulation with IL-2, Vδ3 T cells were expanded to recognize CD1d. Once activa-tion, they can kill CD1d+ target cells, release cytokines such as Th1, Th2, and Th17, and induce maturation of dendritic cells (DCs) into APCs [19].

γδ T cell receptor Since the discovery of γδ T cells more than

twenty five years ago, many articles have been pub-lished to address the physiological functions of these lymphocytes in animals and humans. These studies have emphasized a marked molecular, phenotypic, and functional heterogeneity of γδ T cells. γδ T cells account for 1-10% of CD3+ peripheral blood T lym-phocytes of healthy adults [20] and the ratio of

Vδ2/Vδ1 cells in blood is approximately 3–10. It is thus most likely that environmental or genetic factors control the balance of the subsets [21, 22]. Compen-satory changes for Vδ2 and Vδ1 populations in blood demonstrate a homeostatic balancing mechanism that controls the overall blood count [23]. Caccamo and colleagues showed the influence of gender and age on blood Vδ2 levels, with women having lower levels, compared to age-matched men [24]. These studies suggest that Vδ2 T cell levels be controlled by multi-ple mechanisms, resulting in the difference in pe-ripheral blood γδ T cell populations among people.

The crystal structure of a human Vγ9Vδ2 γδ TCR [25] illustrates that the receptor shares similarities with the heavy and light chains of immunoglobulins, including the formation of a cleft between the CDR3 loops that is similar to the combining site of immu-noglobulin. The amino acid residues consisting of the pocket are mostly germ-line-encoded. This strongly suggests that γδ T cells directly recognize nonpeptide antigens, in a manner similar to antibodies. The recognition of phosphoantigens, however, requires cell–cell contact [26], which indicates that an as yet unidentified molecule(s) is also required to present phosphoantigens to γδ TCR or to transduce the γδ TCR mediated-signaling. Recent reports suggest that ATP synthase-F1/apolipoprotein A-1 complex and butyrophilin 3 are involved in the recognition of nonpeptide antigens by γδ T cells [27, 28].

It is difficult to identify antigens for γδ T cells other than phosphoantigens, because immunization, vaccination, or infection does not generally elicit an-tigen-specific γδ T cells. It is suggested that the γδ TCR repertoire is directed against “self,” most likely represented by inducible cell surface molecules that reflect the status of a cell or tissue [29]. Apart from γδ TCR, γδ cells express most T-lineage-specific genes, including cell surface receptors, signaling effectors, cytokines, and transcription factors, and they pheno-typically and functionally resemble αβ cells in both activated and resting states [30].

Classification of γδ T cells γδ T cells are distinguished from their αβ T cell

counterparts by utilizing a distinct set of somatically rearranged variable (V), diversity (D), joining (J), and constant (C) genes. Compared to αβ T cells, available germ line repertoires of Vγ and Vδ genes are limited. γδ T cells contain fewer V, D, and J segments than αβ T cells. Regarding evolutionary forces that shape the V, D, and J gene segments, distinct forces were in-volved in the formation of the γ and δ loci. In fact, the primate Vγ and Jδ genes are highly conserved, whereas, the γ-locus is split: the Vγ9, Vγ10, and Vγ11 genes represent the conserved region of the Vγ gene


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locus, but the remaining Vγ genes have been evolving rapidly [31].

According to different TCR γδ chain expression, human γδ T cells can be classified into two subsets: Vδ1 and Vδ2 γδ T cells. Vδ1 T cells with different Vγ predominate in the intraepithelial subset of mucosal γδ T cells where the TCRs appear to recognize stress molecules on epithelial cells [32]. Vδ2 T cells that generally coexpress Vγ9 are abundant in the periph-eral blood and lymphatic system. Besides, they can also be classified into four subpopulations based on their expression of CD27 and CD45RA: naive (CD27+CD45RA+), effector memory (CD27-CD45RA-), central memory (CD27+CD45RA-) and terminally dif-ferentiated (CD27-CD45RA+) [33]. These subpopula-tions can exhibit unique functions during mycobacte-rial infection that correspond to the functions of their αβ T cell analogues [33].

Although the number of available Vδ genes is quite small, the combinatorial diversity of the γδ TCR repertoire is at least as large as the αβ TCR repertoire, owing to extensive non-genetically determined mechanisms: for example, N region insertions, im-precise joining, usage of three reading frames, and so on.

Mechanism of ligand recognition γδ T cells can recognize a variety of structurally

different ligands that vary much in size, composition and molecular structure, including non-peptidic an-tigens, MHC and non-MHC cell surface molecules, soluble proteins, sulfatide and so on.

Non-peptidic antigens Pfeffer et al. [34, 35] suggested that non-peptide

antigens may be important targets for T-cell recogni-tion, which may occur for infections with fungus, bacteria, or protozoa. Porcelli et al. [36] demonstrated that monoalkyl phosphates (MEP) might stimulate γδ T cells in a TCR-dependent manner which was similar to naturally occurring non-peptide mycobacterial an-tigens. The natural non-peptide antigens are usually fixed in their structure with less antigenic modulation and are much smaller, so the recognition of these an-tigens is more quickly and effectively with as fast as 2-3 minutes after exposure, indicating that it is not need for antigen uptake and processing. Studies also have confirmed that γδ T cells are specific to recognize phosphate-containing compounds with short, non-bulky carbon chains [37, 38]. Recognition of the prenyl pyrophosphates by the Vγ9Vδ2 γδ TCRs de-pends on all CDRs [39], as these small molecules are usually presented on the surface of target cells. These findings provide convincing evidence that human γδ T cells can recognize a variety of small phosphory-

lated non-peptide antigens. Butyrophilin BTN3A1 has been identified as

phosphorylated antigen-presenting molecule (APM) to Vγ9Vδ2 T cells [40], which belongs to a family of immunoglobulin-like molecules with immunomodu-latory functions [28, 40, 41]. It plays an important role in Vγ9Vδ2 TCR recognition of prenyl pyrophos-phates. Researches have found that it seems function as a sensor for the intracellular levels of phosphory-lated antigens, not the same as a costimulatory ligand or a classical presenting molecule binding prenyl py-rophosphates with its extracellular domains [42]. BTN3A1 molecules are modified via specifically binding with these antigens, thus leaving more free space to facilitate the binding of antigens [40]. The identification of BTN3A1 as an additional APM en-riches the field of human immunological recognition, with potentially far-reaching implications for clinical immunotherapy.

MHC molecules γδ T cells can recognize MHC molecules such as

group 1(CD1a, b, c) and group 2 (CD1d) CD1 mole-cules [43, 44]. The stress-induced MHC class I-related molecules MICA and MICB including α1 and α2 do-mains are also involved in the recognition by human γδ T cells, and do not need Ag processing [45]. Will-cox et al. demonstrated a new kind of EPCR-dependent recognition of cytomegalovirus (CMV)–infected cells by γδ T cells [46]. They found that human γδ TCR could directly bound endothelial protein C receptor (EPCR), which is a MHC–like molecule that binds lipids similar to the anti-gen-presenting molecule CD1d and can make γδ T cells recognize endothelial cells which are targets for cytomegalovirus infection and epithelial tumors. CMV infection can activate γδ T cells specific for self antigens inducing by a multimolecular stress signa-ture on the surface of infected cells, containing EPCR and costimulatory ligand (s). The γδ TCR bound EPCR in an antibody-like way, independently of li-pids. Stress surveillance by γδ T cells may consist of innate-like (NKG2D-mediated) components and adaptive (TCR-specific) components, with the γδ TCR engaging a stress-regulated self antigen.

Soluble proteins Some soluble proteins are also involved in the

recognition by γδ T cells, for example, bacterial pro-teins including the unrelated staphylococcal entero-toxinA (SEA) [47] and the toxin listeriolysin O (LLO) [48]. γδ T cells can also recognize heat shock proteins (HSPs), which does not need antigen processing and can occur in the absence of any APCs [49]. There is no substantial evidence demonstrating that HSP recog-


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nition involves the TCR. HSPs may signal through TLRs, directly or indirectly through binding peptides or hydrophobic ligands, such as LPS [50, 51].

In addition, researches show that the mye-lin-derived glycosphingolipid sulfatide can also be recognized by human γδ T cells [52], indicating that there may also exist other kinds of non-peptidic Ags for γδ T cells which have not yet been identified.

NKG2D, which is expressed on Vγ9Vδ2 T cells, plays an important role in the ligand recognition by γδ T cells, and also is key molecular determinants for the Vγ9Vδ2 T-cell recognition of various carcinomas [53, 54]. NKG2D is a C-type lectin receptor, which provides activation signals by binding to its ligands such as MIC and ULBP families. NKG2D ligands are not expressed by most normal tissues but are upreg-ulated by many tumor-cell types, which are required for tumor cell-recognition by Vγ9Vδ2 T cells.

The precise molecular mechanisms of ligand recognition by γδ T cells remain to be elusive: one supposing that the recognition of nonpeptide antigens by γδ T cells is essentially the same as that by immu-noglobulins (Igs) [55], just binding to the specific lig-and; the other one is the mechanism similar to that of αβ TCRs, involving a specific ligand and a self-identifying presenting molecule [56]. Binding studies exihibit the existence of Ig-like recognition and a crystallographic analysis supports the direct receptor–ligand interaction for proteinaceous antigens [57]. But further investigation has to be conducted to verify the underlying mechanism of the γδ T cell recognition of nonpeptide antigens, because third molecule may possibly be involved in the efficient recognition.

Activation of γδ T cells Early studies demonstrated that most Vγ9Vδ2 T

cells are strongly activated by a variety of killed mi-croorganisms including bacteria such as Mycobacte-rium tuberculosis [58] and parasites such as Plasmodium falciparum [59]. Later, antigens have been identified as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMB-PP), an immediate upstream metabolite of isopentenyl diphosphate (IPP), so called phosphoan-tigens [60]. The phosphoantigens can specifically ac-tivate Vγ9Vδ2 T cell subset at pico- to nanomolar concentrations. The bacterial HMB-PP levels deter-mine the magnitude of Vγ9Vδ2 T cell expansion in vitro or upon transfer of human PBMC and subse-quent infection with bacteria. Analogues with potent and selective stimulatory activity for Vγ9Vδ2 T cells can be synthesized. For instance, bromohydrin di-phosphate (BrHPP) is active at nanomolar concentra-tions [61].

In contrast to many bacteria and protozoas, hu-man cells utilize the mevalonate pathway for isopre-noid and IPP biosynthesis [62, 63], which also acti-vates Vγ9Vδ2 T cells in vitro following presentation by professional antigen-presenting cells or tumor cells [64, 65], but only at concentrations not achieved physiologically in non-transformed cells. However, certain tumors yield higher concentrations of IPP, which can be sensed by Vγ9Vδ2 T cells [66, 67].

The intracellular levels of IPP can be manipu-lated by the therapeutically administered drugs. Ni-trogen-containing bisphosphonates (N-BPs) such as pamidronate (Pam) and zoledronic acid (Zol), which are in clinical use for osteoporosis and hypercalcemia of malignancies, can enhance intracellular levels of IPP by the inhibition of farnesyl diphosphate synthase (FPPS) [68-70], contributing to the activation and ex-pansion of human Vγ9Vδ2 T cells (Fig. 1).

Figure 1. Nonpeptide antigens for γδ T cell stimulation.


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Because the activation of γδ T cells does not re-quire antigen processing or MHC molecules, but re-lies on cell-cell contact with APCs [71], stimulated γδ T cells themselves seem to serve as APCs, but the self-activation or presentation is not effective, com-pared to the optimal stimulation by monocytes or tumor cells [72]. This indicates that TCR recognition of phosphoantigens requires antigen presentation mol-ecules on APCs. In fact, tetramers of human γδ TCRs bind to APCs in an antigen-dependent manner [73-75].

Recently, Harly and coworkers [27] made a sig-nificant advancement on the mechanism underlying the activation of human Vγ9Vδ2 T cells. They found that CD277, a member of butyrophilin molecules, played a central role during the γδ T cell activation. It is, however, still unclear how the Vγ9Vδ2 T cells rec-ognize the phosphoantigen (or anti-CD277 mAb)–induced perturbation of the CD277 surface molecule [76]. The requirement of CD277 for the recognition may explain why human γδ T cells rec-ognize phosphoantigens in a species-specifc manner, because there is no CD277 ortholog in rodents.

In addition, the activation of γδ T cells also re-quires a variety of costimulatory molecules, including immunoglobulin (Ig) superfamily coreceptors (like CD28 or JAML), tumor necrosis factor receptor (like CD27) and atypical costimulatory molecules such as NKG2D or CD46.

Ig superfamily coreceptors Several functional assays have suggested CD28

plays an active role in γδ T cell activation [77, 78], which may produce both qualitative and quantitative changes resulting in lower activation threshold and enhanced T cell activity. Anti-CD28 agonist antibodies can enhance human γδ T cell proliferation [79], while blocking antibodies inhibit it obviously [80].

Junctional adhesion molecule-like protein (JAML) has been considered as a key co-receptor in mouse DETC (express an oligoclonal Vγ5Vδ1 TCR) activation [81], whose costimulation can induce DETC proliferation and the secretion of TNF-α, IFN-γ and IL-2. However, it remains unknown whether JAML plays any role in the costimulation of other (including human) γδ T cell subsets.

Tumor necrosis factor receptor (TNFR) CD27, one of TNFR superfamily co-receptors,

has also been shown important contributions to T cell activation. About 80% of Vγ9Vδ2 T cells express CD27 (TNFRSF7) [82]. Upon activation with PMA and ionomycin, most of CD27+ Vγ9Vδ2 T cells produce IFN-γ with less than 1% is IL-17 [82]. The proliferation of CD27+ Vγ9Vδ2 T cells is sensitive to CD70-CD27

modulation, which provides survival and prolifera-tive signals to control γδ T-cell activation. CD27 sig-nals can activate the non-canonical NF-kB pathway and enhance the expression of anti-apoptotic and cell cycle-related genes [83]. Besides, CD27 costimulation plays important roles in the protection from activation induced cell death (AICD) following phosphoantigen stimulation [82] and the expansion of tumour-specific cytotoxic T lymphocytes (CTLs) [84, 85].

Atypical costimulatory molecules The C-type lectin-like NKG2D receptor plays

critical roles in the activation of T cells. NKG2D shows costimulatory function in γδ T cells, which can en-hance the response of Vγ9Vδ2 T cells upon TCR acti-vation. NKG2D ligation in Vγ9Vδ2 T cells can upreg-ulate the activation marker CD69 independently of TCR stimulation [48]. NKG2D can either directly ac-tivate T cells, as happens for NK cells, or act as a co-receptor to the TCR just as CD8+ T cells do [82]. Upon activation, these NKG2D-expressing T cells can kill tumor [86] or pathogen-infected cells [87].

Furthermore, Vγ9Vδ2 T cells express the BC2 isoform of CD46, which can reduce TNF-α and IFN-γ secretion in HMBPP-stimulated Vγ9Vδ2 T cell cul-tures [88] when signaling through this isoform, indi-cating a novel role for CD46 in regulating the produc-tion of (Th1-like) pro-inflammatory cytokines in hu-man γδ T cells.

Functions of γδ T cells γδ T cells display a variety of functions. They can

produce different kinds of cytokines and chemokines such as interferon-γ (IFN-γ) and tumor necrosis fac-tor-α (TNF-α), and growth factors such as IGF-1. Be-sides, they can regulate immune responses via inter-actions with other cells, for example, they can provide help for B cells and present antigens for αβ T cells. Other important roles are involved in the macrophage recruitment, cytolytic activity and so on. Such main functions are detailed below.

Firstly, γδ T cells can produce a vast variety of cytokines and chemokines to regulate other immune (e.g., Tregs) and non-immune cells. The types and physiological roles of these cytokines, chemokines and others are in detail discussed in the part of the subtitle “Cytokine secretion”.

Secondly, they can provide help for B cells [89]. Caccamo group found that a subset of Vγ9Vδ2+ T cells that expresses CXC-chemokine receptor 5 (CXCR5) which defines follicular B Th (Thf) cells were able to induce significant production of Igs even in the ab-sence of Ag, and provided help for naive B cells to produce IgM, IgG, and IgA Abs, suggesting that they are highly efficient at providing B cell help and play a


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significant regulatory role in humoral immunity [90]. Thirdly, γδ T cells can trigger dendritic cell (DC)

maturation. Vγ9Vδ2 lymphocytes can induce full ac-tivation of immature dendritic cells (iDCs) infected with pathogens, such as Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) [91]. Human γδ T cells in-duced DC maturation [92] via TCR-CD1 [93] and Fas–FasL interactions [94]. Because DCs are uniquely resistant to Fas-induced cell death, Fas-FasL interac-tion can transduce maturation signaling. When hu-man Vγ9Vδ2 T cells were co-cultured with iDCs, they induced increased expression of CD86 and MHC class I on iDCs [95], mainly via γδ T cell-derived TNF-α and IFN-γ. DC maturation induced by LPS was further enhanced by γδ T cells [96].

Fourthly, γδ T cells are involved in the macro-phage recruitment. In listeriosis, γδ T cells seem to control the production of macrophage chemoattract-ant protein I, because it was absent in mice genetically deficient in γδ T cells [97]. A recent investigation of wound healing demonstrated that epidermal γδ T cells could regulate keratinocyte proliferation after wounding [98], and induce hyaluronan production by the epithelial cells [99]. During the subsequent tissue repairing processes, hyaluronan is deposited in the extracellular matrix where it becomes involved in the recruitment of macrophages to the wound lesions. In addition, γδ T cells facilitate cellular differentiation of monocytes and macrophage lineage cells [100], as monocytes differentiate into inflammatory macro-phages during bacterial infections, but fail to undergo maturation when γδ T cells are absent.

Fifthly, γδ T cells exhibit varying degrees of cy-tolytic activity to various kinds of malignancies, such as neuroblastomas [101-103]. Chargui et al. [104] suggested that the expanded γδ T cells which were derived from neuroblastoma patients effectively lysed autologous and allogeneic neuroblastoma cells, and zoledronate or BrHPP could increase the susceptibil-ity of neuroblastoma cells to γδ T cells. The cytolytic activity of γδ T cells could be induced through ap-propriate cellular contact [105, 106] and augmented by exposure to cytokines during ex vivo expansion, for example, IL-15 and/ or IL-18 [68, 107]. So ex vivo expanded γδ T cells can function as a promising tool for anti-malignancy immunotherapy.

Sixthly, γδ T cells produce growth factors that maintain epidermal integrity [98]. γδ T cells in Psori-asis, an immune mediated disease associated with hyperproliferation of keratinocytes and angiogenesis, have been showed to produce growth factors such as IGF-1, VEGF and FGF-2[108]. They can up-regulate IGF-1 production when stimulated with HMB-PP or

PMA/Ionomycin, and produce low amounts of VEGF (<100pg/ml) and FGF-2 (<15 pg/mL) upon activa-tion.

Seventhly, they can present antigens for αβ T cells. It was reported by Brandes et al. that Vδ2+ T cells showed principal characteristics of professional anti-gen-presenting cells such as dendritic cells. After ac-tivation, these cells can rapidly express CC-chemokine receptor 7 (CCR7), which drives their migration to lymph nodes, and upregulate their ex-pression levels of MHC class I and class II molecules and the co-stimulators CD80 and CD86 [109]. They can efficiently process antigens and provide co-stimulatory signals which can strongly induce na-ive αβ T cell proliferation and differentiation. Human Vγ9Vδ2+ T cells can also present antigens to CD4+ T cells and cross-present antigens to CD8+ T cells [109].

Cytokine secretion Activated γδ T cells exhibit broad cytotoxic ac-

tivity against a wide variety of tumor cells, in which they utilize death receptor/ligand (e.g. Fas/Fas-ligand)-dependent and perforin/granzyme- or granulysin-dependent pathways. γδ T cells secrete various cytokines [110] and chemokines including proinflammatory Th1-like cytokines such as IFN-γ and TNF-α, and contribute to reduced survival of autologous tumor cells [111]. Besides, they also pro-duce Th2 cytokines such as IL-4 in the bronchoalveo-lar lavage fluid of patients with allergic asthma [112, 113].

Furthermore, some γδ T cells give rise to partic-ular cytokines such as keratinocyte growth factor and connective tissue growth factor (CTGF), showing important functions in the control of epithelial integ-rity, fibrinogenesis and wound repair. Other γδ T cells also produce antimicrobial peptides such as cathelici-din/LL37, indicating the capacity in the local epithe-lial defense.

In addition, γδ T cells have been reported to se-crete interleukin-10 (IL-10) [114], controlling CD8+ T cell expansion and regulating TNF-α secretion by ac-tivated CD8+ T cells, and IL-17 [115], regulating the expansion and recruitment of neutrophils and mon-ocytes to initiate inflammatory responses. The role of IL-17-producing γδ T cells has been evaluated in var-ious models of infection [116] and autoimmunity. Se-cretion of multiple cytokines and chemokines by ac-tivated γδ cells and their physiological roles are shown in Fig. 2


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Figure 2. Secretion by activated γδ cells of multiple cytokines and their physiological roles.

Applications γδ T-cell based cancer immunotherapy

In 1890, Paul Ehrlich proposed vaccines against cancer, which was the first suggestion using the im-mune system to cope with cancer. In fact, immune deficiency states, such as iatrogenic immune suppres-sion, common variable immunodeficiency (CVID), severe combined immunodeficiency (SCID), and ac-quired immunodeficiency syndrome (AIDS), make patients more susceptible to various kinds of malig-nancies [117, 118]. The importance of immune sur-veillance against tumor emergence and progression was reinforced with the observation of different im-mune deficiency states. Targeting the immune system to combat tumors is a promising therapeutic strategy [119] although progress has been slow and success is limited.

Immunotherapeutic approaches for anti-tumor responses via stimulating the adaptive immune sys-tem rely on major histocompatibility complex (MHC)–restricted αβ T cells. Although significant advances in the adaptive immunotherapies toward tumors have been made and vaccine-based strategies have been improved, αβ T cell-mediated immuno-therapies have met with limited success. Durable re-sponses are scarce, and active immunotherapy has not achieved an established treatment modality. αβ T cells require specific tumor-associated antigens (TAAs)

and suitable costimulatory molecules for activation. Once failure or loss of TAAs, MHC molecules, and/or costimulatory molecules occurs, it will render tumor cells less susceptible to αβ T-cell–mediated cytotoxi-city or induce anergy in specific αβ T cells.

γδ T cells are considered to be a member of in-nate immune cells and exhibit an important role in immune-surveillance and immune defense against tumors, including melanomas [120], leukemias, lym-phomas, neuroblastomas, and other types of carci-nomas [102, 104, 121]. The antitumor activity of γδ T cells has been confirmed by expanding them ex vivo followed by infusion to cancer patients [122, 123]. Recently, in vitro–activated γδ T cells have been shown to target a small number of colon cancer stem cells, which had been demonstrated to be attributable to the failure of conventional therapies. In addition, γδ T cells can kill chemotherapy (imatinib)–resistant chronic myelogenous leukemia lines.

γδ T cells can be utilized for antitumor therapies in an unconventional manner [64], because they ex-hibit a potent MHC-unrestricted lytic activity against a wide variety of tumor cells in vitro. In clinical stud-ies, γδ T cells have been shown to infiltrate into dif-ferent kinds of tumors, including lung carcinomas [124], renal cell carcinomas [125], and breast carcino-mas [126]. It is worthy of note that they have exhibited to specifically respond in vitro to tumors but not to normal cells. Moreover, a high level of γδ T cells was found in disease-free survivors of acute leukemia


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following allogeneic bone marrow transplantation [127]. Clinical evidence in the therapy of human ma-lignancies using γδ T cells stimulated with phospho-antigens or bisphosphonates [128] will further sup-port the antitumor effects of this cell population in vivo.

Human γδ T cells mediate anti-tumor immunity via several distinct pathways, such as the secretion of proinflammatory cytokines and pro-apoptotic mole-cules, and cell to cell contact-dependent lysis through an NK-like pathway or a TCR-dependent pathway [129]. Activated γδ T cells secrete large amounts of cytokines, which act on tumor cells or their microen-vironment [130]. IFN-γ, a major cytokine secreted by activated γδ T cells, has multiple anti-tumor effects including direct inhibition of tumor growth, blocking angiogenesis, or stimulation of macrophages. IFN-γ, therefore, is a crucial cytokine in the γδ T cell–mediated anti-tumor responses.

Many reports demonstrate that γδ T cell clones derived from either tumor sites or blood of cancer patients exhibit potent cytotoxicity against tumor cells ex vivo [20]. In addition, γδ T cells have a unique ca-pacity to present antigens to both CD8 and CD4 cells and potentially elicit strong adaptive anti-tumor re-sponses [131]. Furthermore, γδ T cells can respond to stress stimuli originated from transformed cells and exhibit MHC-unrestricted antigen recognition. Acti-vated γδ T cells can exert an immediate and robust direct cytotoxic effect, and simultaneously induce secondary inflammation which attracts tumor specific effector T cells [132]. Adoptive transfer of large num-ber of activated γδ T cells, either alone or along with CD8+ tumor specific T cells, therefore, can potentially boost the dysfunctional host immune system in many ways.

Several clinical trials including patients with advanced diseases refractory to conventional treat-ments have been performed to test the safety and ef-ficacy of γδ T-cell–based immunotherapy. Conse-quently, up to 50% efficacy has been observed in a most recent clinical trial using phosphostim™ for ex-panding the autologous γδ T cells in patients with metastatic renal cell carcinomas [133].

Dokouhaki and his colleagues [134] have built a xenograft model of human non-small cell lung cancer (NSCLC) to study the in vivo function of γδ T cells, and found that the lung cancer progression was re-markably inhibited after the infusion of γδ T cells in the absence of cytokine co-administration. The results

indicate that the interaction between NKG2D and its ligand (s) may partially contribute to the anti-tumor effect of human γδ T cells. Treatment with expanded and activated γδ T cells for NSCLC is especially promising. Firstly, γδ T cells reside in lung tissues and the number is increased in patients with NSCLC. Secondly, γδ T cells can recognize NSCLC cells in spite of the loss of HLA expression found in 69% of the cases. Thirdly, infusion of ex vivo expanded γδ T cells is feasible in patients with NSCLC [135].

To examine the anti-tumor activity of γδ T cells in breast cancer, a phase I trial was conducted, in which Zol and low-dose IL-2 were administered to 10 advanced metastatic breast cancer patients who were therapeutically terminal [136]. The results suggest that the treatment was well tolerated and promoted the effector maturation of Vγ9Vδ2 T cells in all patients. It is noteworthy that there was a statistically significant linkage between clinical outcome and the number of peripheral Vγ9Vδ2 T cells, as seven patients who failed to sustain Vγ9Vδ2 T cells showed progressive clinical deterioration, while three patients who sus-tained robust peripheral Vγ9Vδ2 cell populations displayed one instance of partial remission and two of stable disease, respectively. Consistent with the earli-er clinical trial in prostate cancer, there was a strong correlation of Vγ9Vδ2 T cell status with reduced car-cinoma progression, and Zol plus low-dose IL-2 pro-vided a novel, safe, and feasible strategy to treat re-fractory patients with advanced breast cancer [136]. It has been reported that the treatment of patients with Vγ9Vδ2 T cells plus Zol could efficiently enhance the lysis of MCF-7 breast tumor cells and PC-3 prostate carcinoma cells in a γδ TCR-dependent manner at the effector to target ratios of 30:1 to 7.5:1 [126].

Recently, Kobayashi and his colleagues have reported a complete remission (CR) in a patient with advanced renal cell carcinomas who underwent 6 monthly cycles of autologous γδ T cell therapy, in which the activation and expansion of γδ T cells were performed in vitro using 2-methyl-3-butenyl diphos-phate plus IL-2, followed by the infusion of the ex-panded γδ T cells, Zol (4 mg) plus low-dose IL-2 (1.4×106 IU) [137]. The clinical responses were associ-ated with a sharp increase in the number of IFN-γ-producing adoptively transferred Vγ9Vδ2 T cells. The CR patient has been disease free for more than 3 years without any additional treatment. Some important clinical trials on γδ T-cell-based cancer immunotherapy are summarized in Table 1.


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Table 1. Examples of the important clinical assays about γδ T-cell-based cancer immunotherapy.

Cancer Immunotherapy

Cancer Type Results Ref.

Treated with zoledronate+IL-2

Malignant melanomas γδ T cells and the cancer’s stage are negatively correlated, result in target cell lysis and death

[120]

Treated with zoledronate+IL-2

Hormone-refractory prostate cancer

Partial remission and stable disease, aggregate increases in γδ T cell numbers

[66]

Pretreated with pamidronate and zoledronate

MCF-7 breast tumor Tumor cells were efficiently lysed, depended on the per-forin-granzyme pathway.

[126]

Bone marrow grafts depleted of αβ T cells

Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML)

Have a significant improvement in disease-free survival, a post-BMT absolute increase in γδ T cells was significantly associated with αβ T-cell depletion.

[127]

Autologous γδ T cells infusion alone or with IL-2

Renal cell carcinoma (metastatic) The maximum-tolerated dose and safety of γδ T cells is 8×109 cells.

[128]

Inoculation without cytokines Non-small cell lung cancer Progression is remarkablely inhibited. [134] In vitro proliferation of γδ T cells with pamidronate/IL-2

Non-Hodgkin lymphoma (re-lapsed and/or refractory) or multiple myeloma

Pamidronate/IL-2 was well tolerated, and no dose-limiting toxicity was observed.

[138]

Autologous γδ T cells

Colon carcinoma Recognition and efficient killing of autologous and alloge-neic CCCL (Colon Carcinoma Cell Lines)

[139]

Treated with zoledronic acid Pancreatic carcinoma Tumor cells treated with zoledronic acid are more vulner-able against γδ T cell attack.

[140]

Applications of γδ T cells to treatment of patients with infectious diseases

Infectious diseases are a serious threat to human health and gradually increase in global morbidity and mortality in recent years. Current strategies to control infections mainly focus on the pathogens themselves, but neglect the host factors which may regulate the progression of the diseases. The frequent appearance of drug resistance in infectious pathogens often leads to costly but ineffective therapy. In addition, the effi-ciency of vaccines inducing adaptive immune re-sponses could be impaired by the rapid immune eva-sion of pathogens through their frequent mutations. In conclusion, innate immune cells that recognize the conserved structural components of pathogens and elicit rapid responses against infections have great potential in anti-infection immunotherapy.

Human γδ T cells are vital components of the innate immune system and play important roles in the early responses to invasive pathogens. Besides, some γδ T cells, such as IL-17-producing γδ T cells, have been demonstrated to be involved in the pathogenesis of transplantation rejection, autoimmune disease [141], inflammatory diseases [142] and allergy [143] in humans. The quality and quantity of human γδ T cells with dynamic variation affects the initiation, progres-sion and prognosis of infectious diseases.

Viral infections Although the mechanism underlying γδ T

cell-mediated immune responses against viruses re-mains to be delineated, their protective roles have been confirmed in some acute and chronic viral infec-

tions. The activation and cytokine secretions of γδ T cells are considered as indicators of early viral infec-tion.

Recently, the beneficial effects of human Vγ9Vδ2 T cells against influenza virus infection have been re-ported. Vγ9Vδ2 T cells can control infection of several strains of influenza viruses, such as pandemic H1N1, human seasonal H1N1, and the avian H5N1 and H9N2 viruses [144]. A study has suggested that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-activated cells had a higher capacity to pro-duce IFN-γ [145]. It is worth noting that IPP-activated γδ T cells also can inhibit seasonal and pandemic H1N1 viruses in a noncytolytic manner, mainly through IFN-γ production [145]. Avian H5N1 and H9N2 viruses can induce higher CCL5 production in Vγ9Vδ2 T cells, which may mediate the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells [145].

In addition, Pam activated human Vγ9Vδ2 T cells could kill influenza virus-infected cells and sup-press viral replication in vitro. Tu et al. [146] demon-strated that Pam-expanded Vγ9Vδ2 T cells by them-selves can control influenza virus infection effectively in vivo. Regarding H1N1 viruses, the inhibition of the virus replication by the Vγ9Vδ2 T cells may rely on direct killing of virus-infected cells and secretion of IFN-γ, but, for H5N1 infection, mainly by direct kill-ing.

The antiviral mechanisms elicited by Vγ9Vδ2 T cells during different viral infections are diverse. For example, human Vγ9Vδ2 T cells can kill Epstein-Barr virus (EBV)–, and herpes simplex virus


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(HSV)–infected target cells in an HLA-unrestricted manner in vitro [147]. IPP–activated Vγ9Vδ2 T cells can kill human immunodeficiency virus (HIV)–infected target cells and inhibit viral replication by releasing certain CCR5 ligand chemokines to block the HIV entry co-receptor CCR5. Furthermore, phos-phoantigen-activated Vγ9Vδ2 T cells can induce noncytolytic inhibition of hepatitis C virus (HCV) replication by IFN-γ secretion [148]. It was revealed that simian γδ T cells could produce β-chemokines, such as macrophage inflammatory protein-1α [MIP1-α], MIP1-β, and RANTES. These factors are known to block virus attachment to the CCR5 co-receptor [149], thus preventing SIV infection.

Furthermore, γδ T cells can induce the matura-tion of dendritic cells (DCs) to promote the estab-lishment of protective adaptive immunity against West Nile virus [150]. The protective roles of γδ T cells have also been confirmed in some chronic infectious diseases. During human cytomegalovirus (HCMV) infection [151], Vδ2- T cells, a minor population of peripheral blood γδ T cells, have been found to ex-pand significantly, showing a potent ‘virus-specific’ cytotoxicity and increased elimination of pathogens [152]. Similarly, γδ T cells in HIV-infected patients have been found to exhibit beneficial roles in control-ling HIV infection [153] through their cell-lytic func-tions and cytokine secretions. γδ T cells are potent effectors in antibody-directed cell cytotoxicity [154], which is important for HIV inhibition [155], although the quantity and quality of γδ T cells are generally decreased with the advancement of HIV infection [156]. γδ T cells also help control the infection caused by Epstein–Barr virus [157] and human hepatitis virus C [158].

More recently, Yin’s group found negative cor-relation between the ratios of Vδ2 T cells to Th17 (IL-17-producing CD4+ T) cells and liver damage in HBV–infected immune-activated patients and pro-vided experimental evidence that Vδ2 T cells sup-pressed Th17 cytokine production through cell con-tact-dependent and IFN-γ-dependent mechanisms [116].

The applications of γδ T cells in the antiviral immunity are summarized in Table 2.

Bacterial infections The discovery that γδ T cells expanded in the

peripheral blood of patients with bacterial infections raised the possibility that the T cell subset can be uti-lized for the control of bacterial infections. Mounting evidence indicates that γδ T cells are of importance in human bacterial infections. Human γδ T cells can recognize HMBPP derived from various bacteria and provoke adaptive immunity in various ways. They

expand during bacterial infections such as tuberculo-sis (mean, 14%), salmonellosis (mean, 18%), tularemia (mean, 31%), brucellosis (mean, 29%), listeriosis (mean, 12%), and ehrlichiosis (mean, 57%). Activated and expanded Vγ2Vδ2+ T cells might directly partic-ipate in antimicrobial immune responses. They rec-ognize HMBPP in a TCR–dependent, MHC–, and CD1–unrestricted manner [6], then kill bacte-ria-infected cells and bacteria.

Tabel 2. Applications of γδT-cell in the antiviral immunity.

Virus Type The Functions of γδ T Cells Ref. Influenza virus Cytotoxic and noncytolytic

antiviral activities [144]

Simian immunodeficiency virus (SIV)

Produce β-chemokines, block virus attachment to the CCR5 co-receptor

[149]

West Nile virus Induce the maturation of den-dritic cells (DCs)

[150]

Human Cytomegalovirus (HCMV)

Have ‘virus-specific’ cytotoxi-city

[151]

Human immunodeficiency virus (HIV)

Cell-lysis and cytokine secretion [153]

Hepatitis C virus (HCV) Mediate non-(MHC)–restricted killing of primary hepatocytes, produce Th1-like cytokine

[158]

Pontiac fever-like disease, which is caused by

Legionella micdadei, was found to be related to a sig-nificant and long-lasting expansion of Vγ9Vδ2 T cells, implying that the subset may also be pathophysio-logically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, patients with the Pontiac fever-like disease showed an early depletion of Vγ9Vδ2 T cells from the circulation, fol-lowed by a sharp increase and subsequently, a slow decline over the next 6 months [159]. The ability of the γδ T cells to secrete IFN-γ and TNF-α seemed to be down-regulated after the acute phase of the disease. These results support the assumption that Vγ9Vδ2 T cells are pathophysiologically important in intracel-lular bacterial infections, including a mild and tran-sient condition such as Pontiac fever.

Moreover, some intracellular bacterial patho-gens, such as Mycobacterium tuberculosis, can specifi-cally expand and activate Vγ9Vδ2 γδ T cells by in-ducing the production of metabolites (e.g., IPP) in infected cells, which strongly demonstrates that γδ T cells are crucially important in infection control [33]. Consistent with this finding, the suppression of γδ T cells by chronic tuberculosis infection can contribute to a disastrous outcome.


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Other pathogen infections γδ T cells not only play protective roles in viral

and bacterial infections, but also control infections by protozoas such as Leishmania [160] and Toxoplasma gondii [161], whereas the γδ T cell-mediated inflam-mation may cause some unwanted destruction of surrounding tissues. Similarly, the protective roles of γδ T cells during malaria infection have been con-firmed in several independent studies [162].

Applications of γδT-cell in autoimmune diseases

In recent years, γδ T cells have been shown roles in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus ery-thematosus (SLE). γδ T cells, by bridging innate and adaptive immunity, may display different functions similar to those of CD4+ T-cell subsets such as CTLs, Th1/Th2 cells, Tregs, Th17 cells, and APCs depending on specific microenvironment [163].

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the limbs, but the path-ogenic mechanism is still unclear. Research has demonstrated that γδ T cells can function as anti-gen-presenting cells and are related with rheumatoid arthritis development. During the development of rheumatoid arthritis, γδ T cells can aggravate immune dysfunction and produce abnormal immune damage by the secretion of cytokines (such as IFN-γ and IL-17) and induction of inflammatory cells to participate in synergistic inflammatory responses [164].

Systemic lupus erythematosus (SLE) is a com-mon autoimmune disease with severe dysregulation of the immune system. Research suggested that the CD27+CD45RA- γδ T cells (a subset constitution of the peripheral blood γδ T cells) were significantly de-creased in SLE patients and the numbers of CD27+CD45RA- γδ T cells was negatively correlated with the SLE disease activity. In inactive SLE patients following glucocorticosteroid and cyclophosphamide treatment, Vδ1 cells were significantly increased. This research group also suggested that CD27+CD25high Vδ1 T cells had immunoregulatory activities through cell-to-cell contact, which could express Foxp3 similar to CD4+Foxp3+. These regulatory γδ T cells decreased in the peripheral blood of active SLE patients could be generated in vitro under the stimulation with anti-TCR γδ in the presence of TGF-β and IL-2 [165]. This find-ing provides a theoretical basis and feasibility for employing γδ Tregs as a potential therapeutic target in autoimmune disease immunotherapy.

In addition, in an experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease multiple sclerosis, γδ T cells had been shown to regulate CNS inflammation and pro-

mote disease recovery through Fas/FasL-induced apoptosis of encephalitogenic T cells [166].

Applications of γδT-cell in allergic diseases Some researches have suggested that γδ T cells

may serve as effectors and immunoregulatory cells in allergic disease. Atopic dermatitis (AD), a chronic relapsing inflammatory disease of the skin, is associ-ated with allergic bronchial asthma. Cairo et al. [167] have observed that the circulating Vγ9Vδ2 T cells were significantly increased in AD patients, which is positively correlated between their expansion and the severity of the disease.

Zhang et al. demonstrated that γδ T cells play a proinflammatory role in the development of ovalbu-min-induced allergic airway inflammation [168]. Svensson et al. indicated that γδ T cells promote al-lergic airway inflammation by enhancing the systemic IgE response and local antibody reactivity without a specific role in the shift of the immune response to-wards Th2 [169]. IL-17+ γδ T cells, belonging to the Vγ4 subset, have been recently shown to downmod-ulate central features of allergic reaction in airway inflammation, including Th2 response and lung eo-sinophilia [143]. When activated, γδ T cells are able to produce a number of cytokines and chemokines, with a unique plasticity to produce Th1, Th2, and Th17 cytokines, contributing to the development and reg-ulation of immune responses [170].

Furthermore, Pawankar et al. have observed an important role for the oligoclonally expanded nasal mucosal gamma delta T cells in the pathogenesis of perennial allergic rhinitis (PAR), with the increase of δ1 T cells and able to produce mainly interleukin such as IL-4, IL-5 and IL-13 [171, 172].

Summary In the previous paragraphs, we have elaborated

on the mechanism of ligand recognition, activation, cytokine secretion, and applications of γδ T cells. To be understood better, we summarized the above-mentioned contents in Fig 3.

Obstacles Clinical applications mentioned above provide

enormous opportunities for accelerating the estab-lishment of novel approaches to disease treatment and control, slowing disease progression, reducing comorbidities, and reducing or modifying require-ments for antiretroviral therapy. People, however, envisage that some specific obstacles have to be overcome for the development of γδ T cell immuno-therapies.

Firstly, clinical trials have demonstrated that repeated administration of phosphoantigens might


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cause anergy, exhaustion or even death of effector γδ T cells. For instance, a macaque study showed that declining responses occurred after repeated BrHPP/IL-2 injections compared to the first treatment [173]. A similar pattern of declining responses was reported for Zol and IL-2 treatment in prostate cancer patients [66]. Thus, therapies targeting γδ T cells produce short-term responses for a long-term, chronic disease. If we can extend the duration of elevated γδ T cell levels and functions following treatment, repeti-tive dosing may not be necessary or the detrimental anergizing effect may not occur. Pauza and his col-leagues [22] defined the protocol for γδ T cell activa-tion, including adding immunomodulators such as rapamycin that increase the yield of the cells and po-tentially modulate the onset of anergy [18].

Secondly, immunostimulatory treatments may elicit significant adverse events (SAEs). For instance, activated γδ T cells will produce proinflammatory cytokines that may cause SAEs. People, therefore, must minimize or manage the potential consequences of immune reconstitution. These potential obstacles, the risk for SAEs and anergy, have to be addressed by definitively controlled human clinical trials.

Thirdly, although the activation of Vγ2Vδ2 T cells can contribute to the rapid acquisition of APC characteristics (γδ T-APCs), dominant Vγ2Vδ2 T-cell subset capable of recognizing microbial phosphoan-tigen exist only in primates. Therefore, current task is

to find an analogue to evaluate the characteristics and clinical potential (including side effects) in murine systems, which may be overcome by the development of a humanized mouse model. This is another current key barrier for the application of γδ T cells based immunotherapy.

Outlooks Although great progress has been made in γδ T

cell-based immunothearpies, many aspects need to be improved in future clinical trials.

Regarding tumor cells, it is necessary to explore the adjuvant effect of Toll-like receptor (TLR) stimu-lation, because in vitro treatment of tumor cells with TLR3 and TLR7 agonists could enhance cytotoxicity of γδ T cells isolated from cancer patients [174]. As for γδ T cells, it will be of great importance to evaluate the clinical effects of synthetic TCR agonists such as phosphostim (BrHPP) and picostim (an analog of HMBPP). Another interesting feature is the use of phosphoantigens combined with therapeutic anti-bodies, as suggested by the improved leukemia and/or lymphoma in vitro killing after co-administration of BrHPP and rituximab [175]. In addition, it is important to evaluate the safety of TCR-independent killing strategy by comparing the NKG2D-mediated cytotoxicity against transformed and healthy tissues by NKG2D+ γδ T cells.

Figure 3. Mechanism underlying γδ T cell recognition of nonpeptide antigens and clinical applications.


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Finally, the identification of biomarkers to pre-dict clinical outcome is crucial for patient selection. A recent study, for example, has identified a panel of 10 genes which encode cell surface proteins that segre-gated ''γδ-susceptible'' from ''γδ-resistant'' hemato-logic tumors [121]. Equivalent markers could be promptly characterized in multiple cancer types, and their predictive value should be accessed in γδ T cell-based clinical trials. The combination of ''suscep-tible'' tumor profiles with improved strategies for γδ T -cell activation in vivo may be the way forward for γδ T cell-based cancer immunotherapy.

γδ T cells are attractive targets for cellular im-munotherapy, but protocols for their therapeutic use need to be optimized. In addition, it is necessary to explore better antigens which help us stimulate γδ T cell expansion in vitro for the preparation of a large number of cells for adoptive cell transfer. Future studies should focus on the possible advantages of combining γδ T cell–based immunotherapy with conventional chemotherapy or other therapeutic ap-proaches, such as antiangiogenic drugs.

Acknowledgments We gratefully acknowledge the support by the

Program for Zhejiang Leading Team of Science and Technology Innovation (2011R50021), Social Devel-opment project of Zhejiang Province (2011C23004) and Zhejiang provincial Natural Science Foundation of China (LY12B02019).

Competing Interests The authors have declared that no competing

interest exists.

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Author biography Dr. Yan-Ling Wu is a profes-

sor in Molecular Immunology and now heads the Cellular and Molec-ular Immunology Research Group. She received Master and Doctoral degrees in Applied Life Science in 2003 and in Medicine Science in 2006, respectively, from Tohoku

University, Japan. After that, she entered to Professor Minato’s group of School of Medicine, Kyoto Univer-sity, Japan, as a senior researcher working in the field of molecular immunology. Her current research fo-cuses on understanding the molecular mechanisms of gene regulation related to diseases by immune inhib-itory receptors. Dr. Wu has published better papers as the first/corresponding author in excellent Journals including Chem & Biol, ChemBioChem, Int J Biol Sci etc. and given oral presentations in international confer-ences.

Yan-Ping Ding received her bachelor’s degree in biological sciences from Zhejiang University of Chinese Traditional Medicine, China. Since graduation, she has been working in Zhejiang Center of Disease Control and Prevention, China. Her research mainly focuses

on the field of Molecular Immunology under the guidance of Prof. Yanling Wu.

Yoshimasa Tanaka received his Ph. D in Hokkaido University Graduate School of Agriculture with a specialization in Enzymology and Biochemistry. After graduation, he continued his research in the field of Immunobiology. Since 2008, he is an associate professor and works

in the Center for Innovation in Immunoregulative Immunology and Therapeutics that belongs to the Kyoto University Graduate School of Medicine.

Li-Wen Shen is a postgraduate majoring in pharmacy. He obtained the Bachelor’s degree in Biological Engineering in 2012 from Shanxi Datong University, China. After that, he entered Lab of Molecular Immunology, Zhejiang Provincial Center for Disease Control and

Prevention, China, working with small molecules to regulate disease-related gene to explore gene-targeted drugs under the direction of Profs Y.-L. Wu and W. Zhang.

Chuan-He Wei received his Bachelor’s degree in 2011 from Jilin Agricultural Science and Technol-ogy College, China. Then, he en-tered Prof. Zhang’s group as a postgraduate of College of Phar-maceutical Science, Zhejiang Uni-versity of Technology, China, his

current research mainly focusing on the field of Mo-lecular Immunology under the direction of Profs W. Zhang and Y.-L. Wu.

Dr. Nagahiro Minato is a full professor of Department of Im-munology and Cell Biology, Graduate School of Medicine, Kyoto University, Japan, since 1992, and since 1998, also a profes-sor of Department of Bio-Regulation, Graduate School of

Biostudies, Kyoto University (Joint appointment), Kyoto, Japan. He received his MD & PhD from Kyoto University in 1975. At present, his research focuses on Immunobiology. Prof Minato has published more than 150 papers in excellent Journals, including Nature Medicine, Nature Cell Biol, Nature Immunol, Nature Med, Proc Natl Acad Sci USA, J Immunol, Blood, J Exp Med etc.


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Dr. Wen Zhang is a full pro-fessor with 30 years of research and teaching experience in Bioorganic Chemistry and Chemical Biology. Dr. Zhang earned his doctorate de-gree in Bioorganic Chemistry from East China University of Science and Technology, China. Then, he

entered Professor Ohrui’s Lab of Tohoku University, Japan, working in the field of molecular recognition as a JSPS postdoctoral fellow. After that, he joined Pro-fessor Sugiyama’s Chemical Biology group of Kyoto University, Japan, as a COE and JST research fellow working on biology and chemistry of polyam-ide-nucleic acids interaction. Now, Dr. Zhang has a special interest in elucidating the gene regulation mechanisms with small organic molecules and the development of gene-targeted drug. His group formed in 2008 and established an extremely fruitful collaboration with Prof. Sugiyama's Group in order to better pursue aspects of gene-targeted drug research. To date, Dr. Zhang has published better papers as the first/corresponding author in excellent Journals in-cluding JACS, ChemBioChem, Int J Biol Sci etc.

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